Did aculeate silk evolve as an antifouling material?

Many of the challenges we currently face as an advanced society have been solved in unique ways by biological systems. One such challenge is developing strategies to avoid microbial infection. Social aculeates (wasps, bees and ants) mitigate the risk of infection to their colonies using a wide range of adaptations and mechanisms. These adaptations and mechanisms are reliant on intricate social structures and are energetically costly for the colony. It seems likely that these species must have had alternative and simpler mechanisms in place to ensure the maintenance of hygienic domicile conditions prior to the evolution of these complex behaviours. Features of the aculeate coiled-coil silk proteins are reminiscent of those of naturally occurring α-helical antimicrobial peptides (AMPs). In this study, we demonstrate that peptides derived from the aculeate silk proteins have antimicrobial activity. We reconstruct the predicted ancestral silk sequences of an aculeate ancestor that predates the evolution of sociality and demonstrate that these ancestral sequences also contained peptides with antimicrobial properties. It is possible that the silks evolved as an antifouling material and facilitated the evolution of sociality. These materials serve as model materials for consideration in future biomaterial development.Funding provided by the Australian Commonwealth Scientific and Industrial Research Organisation. Antimicrobial screening was performed by CO-ADD (The Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and The University of Queensland (Australi

Phosphorescent oxygen-sensing and singlet oxygen production by a biosynthetic silk

A recombinant coiled-coil silk was utilised to immobilise heavy-metal-macrocycles which are known to undergo efficient intersystem crossing from the singlet state to the triplet state following excitation with visible light. This spin-forbidden transition leads to phosphorescence and the production of cytotoxic oxygen species. We explore the requirements for specific binding of these macrocycles and demonstrate that immobilisation does not adversely affect their photochemical properties. The biocompatible materials developed here have potential biomedical applications in photodynamic therapy (PDT) and dynamic oxygen-sensing

Engineering a solid-state metalloprotein hydrogen evolution catalyst

Hydrogen has the potential to play an important role in decarbonising our energy systems. Crucial to achieving this is the ability to produce clean sources of hydrogen using renewable energy sources. Currently platinum is commonly used as a hydrogen evolution catalyst, however, the scarcity and expense of platinum is driving the need to develop non-platinum-based catalysts. Here we report a protein-based hydrogen evolution catalyst based on a recombinant silk protein from honeybees and a metal macrocycle, cobalt protoporphyrin (CoPPIX). We enhanced the hydrogen evolution activity three fold compared to the unmodifed silk protein by varying the coordinating ligands to the metal centre. Finally, to demonstrate the use of our biological catalyst, we built a proton exchange membrane (PEM) water electrolysis cell using CoPPIX-silk as the hydrogen evolution catalyst that is able to produce hydrogen with a 98% Faradaic efciency. This represents an exciting advance towards allowing proteinbased catalysts to be used in electrolysis cells

Confirmation of Bioinformatics Predictions of the Structural Domains in Honeybee Silk

Honeybee larvae produce a silk made up of proteins in predominantly a coiled coil molecular structure. These proteins can be produced in recombinant systems, making them desirable templates for the design of advanced materials. However, the atomic level structure of these proteins is proving difficult to determine: firstly, because coiled coils are difficult to crystalize; and secondly, fibrous proteins crystalize as fibres rather than as discrete protein units. In this study, we synthesised peptides from the central structural domain, as well as the N- and C-terminal domains, of the honeybee silk. We used circular dichroism spectroscopy, infrared spectroscopy, and molecular dynamics to investigate the folding behaviour of the central domain peptides. We found that they folded as predicted by bioinformatics analysis, giving the protein engineer confidence in bioinformatics predictions to guide the design of new functionality into these protein templates. These results, along with the infrared structural analysis of the N- and C-terminal domain peptides and the comparison of peptide film properties with those of the full-length AmelF3 protein, provided significant insight into the structural elements required for honeybee silk protein to form into stable materials

Modification of Honeybee Silk by the Addition of Antimicrobial Agents

Honeybee silk proteins can be produced at high levels in recombinant systems, fabricated into materials, and are tolerant of amino acid modifications: properties that make them exciting templates for designing new functional materials. Here, we explore the properties of materials either made from silk-antimicrobial peptide (AMP) fusion proteins or silk containing entrapped AMPs or silver nanoparticles. Inclusion of AMP within the silk protein sequence did not affect our ability to express the proteins or process them into films. When AMP-silk proteins and Escherichia coli cells were coincubated in solution, a reduction in cell numbers was observed after degradation of the chimeric protein to release a truncated version of the AMP. In films, the AMP was retained in the silk with leaching rates of <1% per day. Films containing silver nanoparticles were antimicrobial, with the silk preventing aggregation of nanoparticles and slowing the rate of dissolution of the particles

Intramolecular electron transfer in sulfite-oxidizing enzymes: Elucidating the role of a conserved active site arginine

A central conserved arginine, first identified as a clinical mutation leading to sulfite oxidase deficiency, is essential for catalytic competency of sulfite oxidizing molybdoenzymes, but the molecular basis for its effects on turnover and substrate affinity have not been fully elucidated. We have used a bacterial sulfite dehydrogenase, SorT, which lacks an internal heme group, but transfers electrons to an external, electron accepting cytochrome, SorU, to investigate the molecular functions of this arginine residue (Arg78). Assay of the SorT Mo centre catalytic competency in the absence of SorU showed that substitutions in the central arginine (R78Q, R78K and R78M mutations) only moderately altered SorT catalytic properties, except for R78M which caused significant reduction in SorT activity. The substitutions also altered the Mo-centre redox potentials (Mo(VI/V) potential lowered by ca. 60-80mV). However, all Arg78 mutations significantly impaired the ability of SorT to transfer electrons to SorU, where activities were reduced 17 to 46-fold compared to SorT(WT), precluding determination of kinetic parameters. This was accompanied by the observation of conformational changes in both the introduced Gln and Lys residues in the crystal structure of the enzymes. Taking into account data collected by others on related SOE mutations we propose that the formation and maintenance of an electron transfer complex between the Mo centre and electron accepting heme groups is the main function of the central arginine, and that the reduced turnover and increases in KMsulfite are caused by the inefficient operation of the oxidative half reaction of the catalytic cycle in enzymes carrying these mutations

FAD-sequestering proteins protect mycobacteria against hypoxic and oxidative stress

The ability to persist in the absence of growth triggered by low oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsq is required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.This work was supported by the Maurice Wilkins Centre for Molecular Biodiscovery and the Marsden Fund, Royal Society, New Zealand. This work was also supported by a University of Otago Doctoral Scholarship and a Sandy Smith Scholarship (to L. K. H.) and by a CSIRO OCE Postdoctoral Fellowship, ARC DECRA Fellowship DE170100310, and NHMRC New Investigator Grant APP1139832 (to C. G.). In addition, this work was supported by NHMRC Project Grant APP1128929 (to C. J. J., C. G., and G. M. C.)

Structural Dynamics of a Lytic Peptide Interacting with a Supported Lipid Bilayer

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ1, due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ2, was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ1 increased, indicating a conformational change of the peptide. The values of τ1 and τ2 indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore

FAD-sequestering proteins protect mycobacteria against hypoxic and oxidative stress

The ability to persist in the absence of growth triggered by low oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsq is required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.</p